A microbial-based cancer vaccine for induction of EGFRvIII-specific CD8+ T cells and anti-tumor immunity.

Dysregulated signaling via the epidermal growth factor receptor (EGFR)-family is believed to contribute to the progression of a diverse array of cancers. The most common variant of EGFR is EGFRvIII, which results from a consistent and tumor-specific in-frame deletion of exons 2-7 of the EGFR gene. This deletion generates a novel glycine at the junction and leads to constitutive ligand-independent activity. This junction forms a novel shared tumor neo-antigen with demonstrated immunogenicity in both mice and humans. A 21-amino acid peptide spanning the junctional region was selected, and then one or five copies of this 21-AA neo-peptide were incorporated into live-attenuated Listeria monocytogenes-based vaccine vector. These vaccine candidates demonstrated efficient secretion of the recombinant protein and potent induction of EGFRvIII-specific CD8+ T cells, which prevented growth of an EGFRvIII-expressing squamous cell carcinoma. These data demonstrate the potency of a novel cancer-specific vaccine candidate that can elicit EGFRvIII-specific cellular immunity, for the purpose of targeting EGFRvIII positive cancers that are resistant to conventional therapies.

Reversible inactivation and desiccation tolerance of silicified viruses.

Long-distance host-independent virus dispersal is poorly understood, especially for viruses found in isolated ecosystems. To demonstrate a possible dispersal mechanism, we show that bacteriophage T4, archaeal virus Sulfolobus spindle-shaped virus Kamchatka, and vaccinia virus are reversibly inactivated by mineralization in silica under conditions similar to volcanic hot springs. In contrast, bacteriophage PRD1 is not silicified. Moreover, silicification provides viruses with remarkable desiccation resistance, which could allow extensive aerial dispersal.

A self-help program for memory CD8+ T cells: positive feedback via CD40-CD40L signaling as a critical determinant of secondary expansion.

The ability of memory CD8+ T cells to rapidly proliferate and acquire cytolytic activity is critical for protective immunity against intracellular pathogens. The signals that control this recall response remain unclear. We show that CD40L production by memory CD8+ T cells themselves is an essential catalyst for secondary expansion when systemic inflammation is limited. Secondary immunization accompanied by high levels of systemic inflammation results in CD8+ T cell secondary expansion independent of CD4+ T cells and CD40-CD40L signaling. Conversely, when the inflammatory response is limited, memory CD8+ T cell secondary expansion requires CD40L-producing cells, and memory CD8+ T cells can provide this signal. These results demonstrate that vaccination regimens differ in their dependence on CD40L-expressing CD8+ T cells for secondary expansion, and propose that CD40L-expression by CD8+ T cells is a fail-safe mechanism that can promote memory CD8+ T cell secondary expansion when inflammation is limited.

Suppression of cell-mediated immunity following recognition of phagosome-confined bacteria.

Listeria monocytogenes is a facultative intracellular pathogen capable of inducing a robust cell-mediated immune response to sub-lethal infection. The capacity of L. monocytogenes to escape from the phagosome and enter the host cell cytosol is paramount for the induction of long-lived CD8 T cell-mediated protective immunity. Here, we show that the impaired T cell response to L. monocytogenes confined within a phagosome is not merely a consequence of inefficient antigen presentation, but is the result of direct suppression of the adaptive response. This suppression limited not only the adaptive response to vacuole-confined L. monocytogenes, but negated the response to bacteria within the cytosol. Co-infection with phagosome-confined and cytosolic L. monocytogenes prevented the generation of acquired immunity and limited expansion of antigen-specific T cells relative to the cytosolic L. monocytogenes strain alone. Bacteria confined to a phagosome suppressed the production of pro-inflammatory cytokines and led to the rapid MyD88-dependent production of IL-10. Blockade of the IL-10 receptor or the absence of MyD88 during primary infection restored protective immunity. Our studies demonstrate that the presence of microbes within a phagosome can directly impact the innate and adaptive immune response by antagonizing the signaling pathways necessary for inflammation and the generation of protective CD8 T cells.

Unanticipated antigens: translation initiation at CUG with leucine.

Major histocompatibility class I molecules display tens of thousands of peptides on the cell surface for immune surveillance by T cells. The peptide repertoire represents virtually all cellular translation products, and can thus reveal a foreign presence inside the cell. These peptides are derived from not only conventional but also cryptic translational reading frames, including some without conventional AUG codons. To define the mechanism that generates these cryptic peptides, we used T cells as probes to analyze the peptides generated in transfected cells. We found that when CUG acts as an alternate initiation codon, it can be decoded as leucine rather than the expected methionine residue. The leucine start does not depend on an internal ribosome entry site-like mRNA structure, and its efficiency is enhanced by the Kozak nucleotide context. Furthermore, ribosomes scan 5' to 3' specifically for the CUG initiation codon in a eukaryotic translation initiation factor 2-independent manner. Because eukaryotic translation initiation factor 2 is frequently targeted to inhibit protein synthesis, this novel translation mechanism allows stressed cells to display antigenic peptides. This initiation mechanism could also be used at non-AUG initiation codons often found in viral transcripts as well as in a growing list of cellular genes.